Role of heparan sulfate as a tissue-specific regulator of FGF-4 and FGF receptor recognition

FGF signaling uses receptor tyrosine kinases that form high-affinity complexes with FGFs and heparan sulfate (HS) proteoglycans at the cell surface. It is hypothesized that assembly of these complexes requires simultaneous recognition of distinct sulfation patterns within the HS chain by FGF and the FGF receptor (FR), suggesting that tissue-specific HS synthesis may regulate FGF signaling. To address this, FGF-2 and FGF-4, and extracellular domain constructs of FR1-IIIc (FR1c) and FR2-IIIc (FR2c), were used to probe for tissue-specific HS in embryonic day 18 mouse embryos. Whereas FGF-2 binds HS ubiquitously, FGF-4 exhibits a restricted pattern, failing to bind HS in the heart and blood vessels and failing to activate signaling in mouse aortic endothelial cells. This suggests that FGF-4 seeks a specific HS sulfation pattern, distinct from that of FGF-2, which is not expressed in most vascular tissues. Additionally, whereas FR2c binds all FGF-4–HS complexes, FR1c fails to bind FGF-4–HS in most tissues, as well as in Raji-S1 cells expressing syndecan-1. Proliferation assays using BaF3 cells expressing either FR1c or FR2c support these results. This suggests that FGF and FR recognition of specific HS sulfation patterns is critical for the activation of FGF signaling, and that synthesis of these patterns is regulated during embryonic development

Activated avb3 Integrin Regulates avb5 Integrin-Mediated Phagocytosis in Trabecular Meshwork Cells

PURPOSE. To investigate the roles of avb3 and avb5 integrins in phagocytosis in human trabecular meshwork (HTM) cells. METHODS. Immunofluorescence microscopy and FACS analysis were used to determine levels of avb3 and avb5 integrins in TM tissue and cultures of normal and immortalized TM cells. Phagocytosis was measured using pHrodo-labeled S. aureus bioparticles followed by FACS analysis. The role of avb5 integrin in phagocytosis was evaluated by knocking down avb5 integrin expression with siRNA against the human b5 gene. Signaling from focal adhesion kinase (FAK) was blocked using FAK inhibitor 14. The role of avb3 integrins in phagocytosis was determined by treating HTM cells with dexamethasone (DEX) or ethanol (EtOH) and by generating stable cell lines that overexpressed either wild type (WT) or constitutively active (CA) b3 integrin subunit. RESULTS. Both TM tissue and cell lines expressed avb3 and avb5 integrins. Knockdown of avb5 integrin reduced phagocytosis by~60% and FAK inhibition significantly reduced phagocytosis up to 84%, in a dose-dependent manner. DEX treatment increased avb3 integrin expression in HTM cells but reduced phagocytosis by~50% compared with untreated and EtOH-treated cells. The CA b3 integrin-expressing cell line showed increased avb3 integrin levels and decreased phagocytosis by~50% compared with the control. CONCLUSIONS. The avb5 integrin-FAK-mediated pathway regulates phagocytosis in TM cells and this pathway is inhibited by activation of avb3 integrins. This suggests that changes in integrin expression and activity may be responsible for alterations in phagocytosis observed in steroid induced glaucoma

Friedreich ataxia patient tissues exhibit increased 5-hydroxymethylcytosine modification and decreased CTCF binding at the FXN locus

© 2013 Al-Mahdawi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,distribution, and reproduction in any medium, provided the original author and source are credited.This article has been made available through the Brunel Open Access Publishing Fund.Friedreich ataxia (FRDA) is caused by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene, which induces epigenetic changes and FXN gene silencing. Bisulfite sequencing studies have identified 5-methylcytosine (5 mC) DNA methylation as one of the epigenetic changes that may be involved in this process. However, analysis of samples by bisulfite sequencing is a time-consuming procedure. In addition, it has recently been shown that 5-hydroxymethylcytosine (5 hmC) is also present in mammalian DNA, and bisulfite sequencing cannot distinguish between 5 hmC and 5 mC.The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number 242193/EFACTS (CS), the Wellcome Trust [089757] (SA) and Ataxia UK (RMP) to MAP

Pharmacological screening using an FXN-EGFP cellular genomic reporter assay for the therapy of Friedreich ataxia

Copyright @ 2013 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol.Muscular Dystrophy Association (USA), the National Health and Medical Research Council (Australia), the Friedreich’s Ataxia Research Alliance (USA), the Brockhoff Foundation (Australia), the Friedreich Ataxia Research Association (Australasia), Seek A Miracle (USA) and the Victorian Government’s Operational Infrastructure Support Program

Generation and characterisation of Friedreich ataxia YG8R mouse fibroblast and neural stem cell models

This article has been made available through the Brunel Open Access Publishing Fund.Background: Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by GAA repeat expansion in the first intron of the FXN gene, which encodes frataxin, an essential mitochondrial protein. To further characterise the molecular abnormalities associated with FRDA pathogenesis and to hasten drug screening, the development and use of animal and cellular models is considered essential. Studies of lower organisms have already contributed to understanding FRDA disease pathology, but mammalian cells are more related to FRDA patient cells in physiological terms. Methodology/Principal Findings: We have generated fibroblast cells and neural stem cells (NSCs) from control Y47R mice (9 GAA repeats) and GAA repeat expansion YG8R mice (190+120 GAA repeats). We then differentiated the NSCs in to neurons, oligodendrocytes and astrocytes as confirmed by immunocytochemical analysis of cell specific markers. The three YG8R mouse cell types (fibroblasts, NSCs and differentiated NSCs) exhibit GAA repeat stability, together with reduced expression of frataxin and reduced aconitase activity compared to control Y47R cells. Furthermore, YG8R cells also show increased sensitivity to oxidative stress and downregulation of Pgc-1α and antioxidant gene expression levels, especially Sod2. We also analysed various DNA mismatch repair (MMR) gene expression levels and found that YG8R cells displayed significant reduction in expression of several MMR genes, which may contribute to the GAA repeat stability. Conclusions/Significance: We describe the first fibroblast and NSC models from YG8R FRDA mice and we confirm that the NSCs can be differentiated into neurons and glia. These novel FRDA mouse cell models, which exhibit a FRDA-like cellular and molecular phenotype, will be valuable resources to further study FRDA molecular pathogenesis. They will also provide very useful tools for preclinical testing of frataxin-increasing compounds for FRDA drug therapy, for gene therapy, and as a source of cells for cell therapy testing in FRDA mice. © 2014 Sandi et al

The GAA triplet-repeat is unstable in the context of the human FXN locus and displays age-dependent expansions in cerebellum and DRG in a transgenic mouse model

Friedreich ataxia (FRDA) is caused by homozygosity for FXN alleles containing an expanded GAA triplet-repeat (GAA-TR) sequence. This expanded GAA-TR sequence is unstable in somatic cells of FRDA patients, showing age-dependent expansions in dorsal root ganglia (DRG), the tissue where pathology occurs earliest and is most significant. This is thought to be the basis for the progressive, tissue-specific pathology seen in FRDA, but the mechanism(s) for this somatic instability is unknown. We show that transgenic mice containing the expanded GAA-TR sequence (190 or 82 triplets) in the context of the human FXN locus show tissue-specific and age-dependent somatic instability that mimics the human condition. Small pool PCR analysis, which allows quantitative analysis of instability by assaying individual transgenes in vivo, showed age-dependent expansions specifically in the cerebellum and DRG. The (GAA)190 allele showed some instability by 2 months, progressed at about 0.3 – 0.4 triplets/week, resulting in a significant number of expansions by 12 months. Repeat length determined the age of onset of somatic instability, and the rate and magnitude of expansion. Whereas the GAA-TR was unstable in the context of the human FXN locus, pure GAATR sequences at other genetic loci in the human and murine genomes showed no instability. These data indicate that somatic instability of the GAA-TR sequence in the human FXN gene is determined by a combination of unique cis and trans-acting factors. This mouse model will serve as a useful tool to delineate the mechanism(s) of diseasespecific somatic instability in FRDA

Role of Fibronectin in Primary Open Angle Glaucoma

Primary open angle glaucoma (POAG) is the most common form of glaucoma and the 2nd most common cause of irreversible vision loss in the United States. Nearly 67 million people have the disease worldwide including >3 million in the United States. A major risk factor for POAG is an elevation in intraocular pressure (IOP). The increase in IOP is believed to be caused by an increase in the deposition of extracellular matrix proteins, in particular fibronectin, in a region of the eye known as the trabecular meshwork (TM). How fibronectin contributes to the increase in IOP is not well understood. The increased density of fibronectin fibrils is thought to increase IOP by altering the compliance of the trabecular meshwork. Recent studies, however, also suggest that the composition and organization of fibronectin fibrils would affect IOP by changing the cell-matrix signaling events that control the functional properties of the cells in the trabecular meshwork. In this article, we will discuss how changes in the properties of fibronectin and fibronectin fibrils could contribute to the regulation of IOP

NFATc1 Regulation of Dexamethasone-Induced <i>TGFB2</i> Expression Is Cell Cycle Dependent in Trabecular Meshwork Cells

Although elevated TGFβ2 levels appear to be a causative factor in glaucoma pathogenesis, little is known about how TGFβ2 expression is regulated in the trabecular meshwork (TM). Here, we investigated if activation of the cytokine regulator NFATc1 controlled transcription of TGFβ2 in human TM cells by using dexamethasone (DEX) to induce NFATc1 activity. The study used both proliferating and cell cycle arrested quiescent cells. Cell cycle arrest was achieved by either cell–cell contact inhibition or serum starvation. β-catenin staining and p21 and Ki-67 nuclear labeling were used to verify the formation of cell–cell contacts and activity of the cell cycle. NFATc1 inhibitors cyclosporine A (CsA) or 11R-VIVIT were used to determine the role of NFATc1. mRNA levels were determined by RT-qPCR. DEX increased TGFβ2 mRNA expression by 3.5-fold in proliferating cells but not in quiescent cells or serum-starved cells, and both CsA and 11R-VIVIT inhibited this increase. In contrast, the expression of other DEX/NFATc1-induced mRNAs (myocilin and β3 integrin) occurred regardless of the proliferative state of the cells. These studies show that NAFTc1 regulates TGFβ2 transcription in TM cells and reveals a previously unknown connection between the TM cell cycle and modulation of gene expression by NFATc1 and/or DEX in TM cells